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研究生:吳仁佑
研究生(外文):Wu Ren-Yu
論文名稱:海鱺淋巴囊腫瘤病毒之檢測及與其它虹彩病毒之親緣關係
論文名稱(外文):Detection of Cobia(Rachycentron canadum)Lymphocystis Disease Virus and Its Phylogenetic Relationship with Other Iridoviruses
指導教授:施秀惠盧秀琴盧秀琴引用關係
學位類別:碩士
校院名稱:國立台北師範學院
系所名稱:數理教育研究所
學門:教育學門
學類:普通科目教育學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:74
中文關鍵詞:海鱺淋巴囊腫病毒巢式聚合酶鏈鎖反應點墨雜交法海鱺鰭細胞株虹彩病毒
外文關鍵詞:Cobia (Rachycentron canadum)Lymphocystis disease iridovirus (LCDV)Nested-PCRdot blot hybridizationCobia fin cell lineIridovirus
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本實驗自屏東小琉球採集某家養殖業者箱網網中的罹病海鱺(Rachycentron canadum),外觀上,鰭與部分體表出現結節狀之小顆粒,嚴重時,呈現瘤狀肉柄,取其各部位組織包括:肝臟、脾臟、腎臟、鰭、心臟、胃、腸等進行固定、切片及染色,發現唯有於鰭與皮膚部位之結締組織細胞,明顯可見肥大之現象,肥大之細胞擁有巨大細胞核與圍繞著細胞核的包涵體網狀結構,由於肥大細胞之堆疊,導致魚體之鰭與一些表皮部位有顆粒狀的「結節」存在。嘗試由肥大之表皮細胞分離病源,並試著感染自己建立之海鱺鰭細胞株(Cobia fin, CF),CF 細胞之初級培養順利、良好,大約每隔 3~4 天即需繼代一次,至今已繼代達 30 多代,外型大致分為纖維狀細胞、小型纖維狀細胞與表皮樣細胞,結果發現除實驗組細胞型態改變外,並無明顯的細胞分解病變現象發生,因此放棄藉由 CF 細胞增殖病毒以供分析。 對於偵測病毒方面,為了增加偵測之便利性與準確度、靈敏度,我們引用 Nested-PCR(巢式聚合酶鏈鎖反應) 與 DIG-labelled dot blot hybridization(點墨雜交法)技術作為偵測方法並比較其優劣程度,結果發現前者之靈敏度遠大於後者,但卻需要較繁重之儀器設備,而後者卻可藉由呈色結果直接判定該組織病情之嚴重性。另一方面,為了瞭解引起海鱺病變之病毒究竟屬於何種病毒,我們透過聚合酶鏈鎖反應(PCR)增幅其主要鞘蛋白(MCP)基因並加以定序,與其他九種已發表的虹彩病毒之 MCP 核酸及胺基酸序列,包括:CzIV、TIV、CIV、LCDV-1、LCDV-K1、BIV、FV3、GIV、RSIV 之 MCP 進行分析比對,顯示 Cobia iridovirus(CbIV)親緣性和感染大多數硬骨魚類的 Lymphocystivirus 屬的 LCDV-(K)1 最為接近(89﹪相似度),和感染兩棲類的 Ranavirus 屬的 FV3 親緣性較低(47﹪相似度)。
Diseased cobias were collected from some net-cage farms at Hsiqu Liouchiou Island, next to Dunggang, Taiwan. Some little nodes appeared on surfaces of their fins and skins. When the symptom became serious, they looked like sarcomas. Almost every organ, including liver, spleen, kidney, fin, heart, stomach, intestine of a hard-sick cobia were dealt through the following processes:fixation, section and staining. Only at the connective tissues of fins and epithelium did we find hypertrophy cells which own big nuclei and inclusion bodies around the nuclei. Owing to the piles of the cells, some nodes appeared on fins and some places of epithelium.We tried to isolate the pathogenic organisms and infect the cells primarily cultured from cobia fins, named CF. The CF cell line was well cultured and passaged every 3~4 days. It had been passaged for over 30 times and owned three types─fibroblast like, little-fibroblast like and epithelium like. To get the opposite of what one wants, the CF failed to get infected with apparent CPE but changed it shape. So that we forgave making use of the CF to proliferate the virus in order to provide our research. As the ways to detect the virus, we compared Nested-PCR with DIG-labelled dot blot hybridization and hoped to rise their degree of accuracy, sensitivity and convenience. The answer was that the former could be far sensitive than the latter, but the former needed complex instruments. The latter could help us to directly judge the order of severity by colors.On the other hand, we took advantage of polymerase chain reaction(PCR)to amplify the MCP genome of the virus and compare it with other nine published MCP genomes of viruses including CzIV, TIV, CIV, LCDV-1, LCDV-K1, BIV, FV3, GIV, RSIV. The sequencing data showed that the MCP of Cobia iridovirus(CbIV)was more homologous to the MCP of LCDV-(K)1(89﹪similarity)than that from FV3(47﹪similarity).
謝誌………………………………………………..Ⅰ
目錄……………………………………………… .Ⅱ
圖表目次……………………………………………Ⅳ
摘要…………………………………………………ⅤAbstract……………………………………………Ⅵ
第一章 研究背景…………………………………1
1.1 海鱺與台灣養殖概況之簡介………………… 1
1.2 虹彩病毒之特性與分類……………………… 3
1.3 虹彩病毒主要鞘蛋白之探討……………… 5
1.4 海鱺鰭細胞株建立…………………………….6
1.5 PCR(聚合酶鏈鎖反應)、Nested-PCR(巢式聚合酶鏈鎖反應)、Dot blot hybridization(點墨雜交法)……….7
1.6 研究動機與目的……………………………….8
第二章 材料與方法…………….……………..10
2.1 海鱺病魚之採樣……………… …………….10
2.2 海鱺之組織切片與染色………….………….10
2.2.1 魚鰭與鰓之脫鈣法…………………………10
2.2.2 組織之脫水與蠟包埋法……………………11
2.2.3 組織切片與染色…………………….… …12
2.3 海鱺鰭細胞株之建立與應用… …………...14
2.3.1 海鱺初級細胞培養……………..........14
2.3.2 細胞之繼代培養……………………………15
2.3.3 細胞之冷凍與解凍…………………………16
2.4 海鱺魚鰭細胞對 CbIV 之感受性試驗………17
2.5 CbIV嵌入海鱺魚體內之基因片段之分析及偵測……..18 2.5.1 感染 CbIV 病魚各組織核酸之萃取…………….….18 2.5.2 利用聚合酶鏈鎖反應(PCR)檢測海鱺各組織中CbIV 主要 鞘蛋白基因部分片段……………………… 19
2.5.3 Nested-PCR 增幅 CbIV 主要鞘蛋白基因部分片段….21 2.5.4 以 PCR 法製備 probe……………………………….… 22 2.5.5 點墨雜交法(Dot blot hybridization)偵
測病毒 MCP 之基因........................23
2.5.6 MCP 之定序………...…………… ……24
2.5.7 MCP 核酸及胺基酸序列之比對…………25
第三章 結果……………………………………26
3.1 罹病海鱺魚體之病灶觀察………………….26
3.2罹病海鱺之組織病理切片觀察………………26
3.3海鱺細胞之初級培養…………………………26
3.4 CbIV 對海鱺鰭細胞株之感受性試驗………27
3.5 以 PCR 法檢測病魚組織中 CbIV 主要鞘蛋白之基因…… 27 3.6 以 Nested-PCR 法檢測病魚組織中 CbIV 主要鞘蛋白之基因…27 3.7以點墨雜交法(Dot blot hybridization)
偵測 CbIV 主要鞘蛋白…...................28
3.8 CbIV 主要鞘蛋白基因之定序………………28
第四章 討論……………………………………30
參考文獻………………………………………… 37
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